1. Field of the Invention
The present invention concerns novel porcine DC-SIGN and porcine LSECtin genes, cDNA derived from the respective porcine monocyte-derived dendritic and liver cells, porcine DC-SIGN and porcine LSECtin proteins, transfected cells or cell lines stably expressing the new proteins, fusion products, antibodies, methods for isolating and cloning the porcine genes and the use of the porcine proteins for propagating viruses. Also provided is the nucleotide sequence encoding newly discovered porcine ICAM-3 isoforms from porcine monocyte-derived dendritic cells.
2. Description of Related Art
All patents and publications cited in this specification are hereby incorporated by reference in their entirety.
Dendritic cells (DCs) are professional antigen-presenting cells (APCs) located throughout the peripheral immune system. Invading foreign antigens trigger the migration of immature DCs from the blood into tissues where they detect and capture the antigens (K. Palucka and J. Banchereau, “Dendritic cells: a link between innate and adaptive immunity,” J. Clin. Immunol. 19:12-25 (1999)). Activated DCs process captured proteins into immunogenic peptides through MHC molecules (a set of membrane glycoproteins called the MHC molecules or the Major Histocompatibility Complex) and present to T cells. Recognition of invading pathogens by DCs is mediated by pattern-recognition receptors (PRRs) including Toll-like receptors (TLRs) and lectins (S. Thoma-Uszynski et al., “Induction of direct antimicrobial activity through mammalian toll-like receptors,” Science 291:1544-1547 (2001); W. I. Weis et al., “The C-type lectin superfamily in the immune system,” Immunol. Rev. 163:19-34 (1998)). The lectins expressed on the surface of DCs are members of the calcium-dependent C-type lectin receptor (CLRs) family and play a key role in the antigen capture and internalization of DCs (Weis et al., 1998, supra). CLRs are also expressed on other APCs including macrophages.
The CLR family includes a large number of proteins that perform protein-carbohydrate interactions by binding to the polysaccharide chains on glycoprotein ligands in a calcium-dependent manner. Numerous CLRs belong to PRRs expressed on the surface of APCs that recognize foreign pathogens, playing a key role in host immune responses. The type II CLRs are classified by their NH2 terminus domain, cytoplasmic tail (CT), located in the cytoplasm of the cell. Other type II CLR domains include the transmembrane domain (TMD) following the CT, a single carbohydrate recognition domain (CRD) at the carboxyl terminus exposed extracellularly and the neck domain between the TMD and CRD.
A human lectin gene cluster of type II CLRs, CD23/LSECtin/DC-SIGN/L-SIGN, which is localized at human chromosome 19p13.3, has received increasing interest. Human DC-SIGN, hL-SIGN and hLSECtin, which have analogous genomic structures (W. Liu et al., “Characterization of a novel C-type lectin-like gene, LSECtin: demonstration of carbohydrate binding and expression in sinusoidal endothelial cells of liver and lymph node,” J. Biol. Chem. 279:18748-58 (2004)), are important C-type lectins capable of mediating pathogen recognition. Human CD23 (FCER2) is a low affinity IgE receptor that plays an important role in cell-cell adhesions, B cells survival and antigen presentation. Dendritic cells-specific intercellular-adhesion-molecule-3 (“ICAM-3”)-grabbing nonintegrin (human CD209, also known as “DC-SIGN,” a 44 kDa type II transmembrane protein), a CLR, was identified as an ICAM-3 binding protein mediating DCs and T cell interaction (T. B. Geijtenbeek et al., “Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3 receptor that supports primary immune responses,” Cell 100:575-585 (2000)) and a HIV-1 gp120 receptor mediating transmission of HIV-1 to susceptible cells in trans (T. B. Geijtenbeek et al, “DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells,” Cell 100:587-597 (2000)). Additionally, DC-SIGN was found to interact with ICAM-2 binding protein, regulating chemokine-induced trafficking of DCs across both resting and activated endothelium (T. B. Geijtenbeek et al., “DC-SIGN-ICAM-2 interaction mediates dendritic cell trafficking,” Nat. Immunol. 1:353-357 (2000)). A second human DC-SIGN (hDC-SIGN) homologue, hL-SIGN (CD209L) or DC-SIGNR, was subsequently identified and shown to have similar function, but subtly distinct property of pathogen recognition, to hDC-SIGN (A. A. Bashirova et al., “A dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN)-related protein is highly expressed on human liver sinusoidal endothelial cells and promotes HIV-1 infection,” J. Exp. Med. 193:671-678 (2001)).
Human DC-SIGN is expressed mainly on monocyte-derived human DCs in vitro, on immature and mature DCs in the normal human lymph node, dermis, mucosa and spleen and on macrophages in alveoli of the lung in vivo (T. B. Geijtenbeek et al., “Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3 receptor that supports primary immune responses,” Cell 100:575-585 (2000); T. B. Geijtenbeek et al., “DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells,” Cell 100:587-597 (2000); L. Tailleux et al., “DC-SIGN induction in alveolar macrophages defines privileged target host cells for mycobacteria in patients with tuberculosis,” PLoS Med. 2:e381 (2005); E. J. Soilleux et al., “Constitutive and induced expression of DC-SIGN on dendritic cell and macrophage subpopulations in situ and in vitro,” J. Leukoc. Biol. 71:445-457 (2002)), whereas L-SIGN is highly expressed in sinusoidal endothelial cells of the liver and lymph node (Bashirova et al., 2001, supra). It has been observed that L-SIGN homologues only exist in human and non-human primates but not in other non-primates mammalian species.
Recently, a third human DC-SIGN-related C-type lectin (identified as “CLEC4G” and named “LSECtin”), which is co-expressed with hL-SIGN on liver and lymph node sinusoidal endothelial cells (LSECs), was identified with similar property of pathogen recognition and antigen capture (A. Dominguez-Soto et al., “The DC-SIGN-related lectin LSECtin mediates antigen capture and pathogen binding by human myeloid cells,” Blood 109:5337-45 (2007)). Besides hLSECtin, LSECtin homologues in other mammalian species have not been experimentally identified although limited gene information can be searched from the genome databases.
Due to similarities in organ size and physiology with humans, pig is considered to be the preferred source animal for xenotransplantation (Y. G. Yang and M. Sykes, “Xenotransplantation: current status and a perspective on the future,” Nat. Rev. Immunol. 7:519-31 (2007)). Understanding the compatibilities across the human-pig species barrier of the molecular interactions is very critical for the clinical application of pig-to-human xenotransplantation. Interactions of the receptors on porcine hematopoietic cells with ligands on human endothelial cells play a crucial role in the event that porcine hematopoietic cells are used to induce tolerance in the human recipient (A. N. Warrens et al., “Human-porcine receptor-ligand compatibility within the immune system: relevance for xenotransplantation,” Xenotransplantation 6:75-8 (1999)). T-cell-mediated xenograft rejection, a phenomenon probably caused by induction of stronger human T cell responses against pig antigen than that against alloantigens, also involved potential interactions of adhesion molecules between porcine APCs such as DCs and human T cells (A. Dorling et al., “Detection of primary direct and indirect human anti-porcine T cell responses using a porcine dendritic cell population,” Eur. J. Immunol. 26:1378-87 (1996)). DC-SIGN has been further shown as the endogenous adhesion receptor for ICAM-2 and ICAM-3 (T. B. Geijtenbeek et al., “Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3 receptor that supports primary immune responses,” Cell 100:575-585 (2000)); T. B. Geijtenbeek et al., “DC-SIGN-ICAM-2 interaction mediates dendritic cell trafficking,” Nat. Immunol. 1:353-357 (2000); D. A. Bleijs et al., “DC-SIGN and LFA-1: a battle for ligand,” Trends Immunol. 22:457-63 (2001)).
Porcine reproductive and respiratory syndrome virus (PRRSV), an economically important swine pathogen worldwide, is a member of the family Arteriviridae in the order of the Nidovirales. PRRSV isolates identified thus far worldwide are divided into two distinct genotypes, European (type 1) and North American (type 2) genotypes, which cause the same disease symptoms but are antigenically different. Like other enveloped viruses such as HIV and HCV, the entry of PRRSV into the host cells, namely, the porcine alveolar macrophages, is a complex multistep process that involves the presence of several entry factors including sialoadhesin, CD163 and heparan sulphate (P. L. Delputte et al., “Analysis of porcine reproductive and respiratory syndrome virus attachment and internalization: distinctive roles for heparan sulphate and sialoadhesin,” J. Gen. Virol. 86:1441-5 (2005)). However, the potential interaction between PRRSV and porcine PRRs on APCs has not yet been reported. Since human L-SIGN was shown to be associated with SARS-coronavirus entry in lung, the porcine DC-SIGN/L-SIGN homologue may play a similar role during PRRSV infection in pig lung since PRRSV and coronavirus both belong to the Nidovirales order but significant experimentation is warranted before a conclusion can be drawn.
Although the monkey kidney cell line (as described in U.S. Pat. No. 6,146,873 and elsewhere) and primary porcine alveolar macrophages (PAMs) have been the only two cells known to support productive PRRSV replication, other cells such as the BHK-21 cell line have been shown to be replication-competent, that is, having the necessary ability to support PRRSV replication (H. Nielsen et al., “Generation of an infectious clone of VR-2332, a highly virulent North American-type isolate of porcine reproductive and respiratory syndrome virus,” J. Virol. 77:3702-11 (2003); J. J. Meulenberg et al., “Infectious transcripts from cloned genome-length cDNA of porcine reproductive and respiratory syndrome virus,” J. Virol. 72:380-7 (1998)). For example, when BHK cells were transfected with viral RNA or in vitro synthesized RNA transcripts from full-length genomic cDNA of European strain LV or North American strain VR-2332, evidence of PRRSV replication was detected in BHK cells. PRRSV virions were produced and excreted into the medium; and when the supernatant from transfected BHK-21 cells was transferred to PRRSV-permissive cells, cythopathic effects (CPE) was observed. Unfortunately, the replicating virus in transfected BHK-21 cells does not spread from cell-to-cell, indicating the lack of receptors on BHK-21 cells. A putative PRRSV binding receptor was reportedly identified from alveolar macrophages to be 210-kDa membrane protein (E. H. Wissink et al., “Identification of porcine alveolar macrophage glycoproteins involved in infection of porcine respiratory and reproductive syndrome virus,” Arch. Virol. 148:177-87 (2003)) but functional confirmation of this receptor candidate at the level of virus entry is still lacking Recently, it has been shown that porcine sialoadhesin (pSn) mediates internalization of PRRSV in PAMs (N. Vanderheijden et al., “Involvement of sialoadhesin in entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages,” J. Virol. 77(15):8207-15 (2003)), and that pSn is a sialic acid binding lectin and interactions between sialic acid on the PRRS virion and pSn are essential for PRRSV infection of PAMs (P. L. Delputte and H. J. Nauwynck, “Porcine arterivirus infection of alveolar macrophages is mediated by sialic acid on the virus,” J. Virol. 78(15):8094-101 (2004)). In human, mice and swine, sialoadhesin is only expressed on discrete subsets of tissue macrophages. PRRSV is known to infect macrophages in the respiratory and lymphoid systems of the pig in vivo. Since PRRSV also infects other monocyte-derived lymphocytes in vivo such as dendritic cells and since the structure of PRRSV virion is very complex, it is likely that multiple alternative receptors and/or co-receptors exist on these cells. In addition, PPRSV receptor on the susceptible monkey kidney cells has not yet been identified.
Macrophages and dendritic cells are important for recognition of pathogens and play important roles in immunity against invading pathogens. Human DC-SIGN and the related liver endothelial cell lectin L-SIGN have been characterized and found to express abundantly on the surface of dendritic-like cells (A. Puig-Kroger et al., “Regulated expression of the pathogen receptor dendritic cell-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin in THP-1 human leukemic cells, monocytes, and macrophages,” J. Biol. Chem. 279(24):25680-8 (2004)). Furthermore, the C-type mannose binding lectins hDC-SIGN and hL-SIGN (or DC-SIGNR) have generated considerable interest for their ability to bind and uptake pathogens including enveloped viruses such as HIV, bacteria (Mycobacterium), fungi and parasites in vitro (Y. van Kooyk and T. B. Geijtenbeek, “DC-SIGN: escape mechanism for pathogens,” Nat. Rev. Immunol. 3:697-709 (2003)), Dengue virus (E. Navarro-Sanchez et al., “Dendritic-cell-specific ICAM3-grabbing non-integrin is essential for the productive infection of human dendritic cells by mosquito-cell-derived dengue viruses,” EMBO Rep. 4(7):723-8 (2003)), Ebola virus (C. P. Alvarez et al., “C-type lectins DC-SIGN and L-SIGN mediate cellular entry by Ebola virus in cis and in trans,” J. Virol. 76(13):6841-4 (2002)), Marburg virus (A. Marzi et al., “DC-SIGN and DC-SIGNR interact with the glycoprotein of Marburg virus and the S protein of severe acute respiratory syndrome coronavirus,” J. Virol. 78(21):12090-5 (2004)), SARS-coronavirus (id.), cytomegalovirus (F. Halary et al., “Human cytomegalovirus binding to DC-SIGN is required for dendritic cell infection and target cell trans-infection,” Immunity 17(5):653-64 (2002)), and hepatitis C virus (P. Y. Lozach et al., “C-type lectins L-SIGN and DC-SIGN capture and transmit infectious hepatitis C virus pseudotype particles,” J. Biol. Chem. 279(31):32035-45 (2004); E. G. Cormier et al., “L-SIGN (CD209L) and DC-SIGN (CD209) mediate transinfection of liver cells by hepatitis C virus,” Proc. Natl. Acad. Sci. USA 101:14067-72 (2004)) to facilitate entry into cells and infection. Both hDC-SIGN and hL-SIGN contain C-type-lectin-specific carbohydrate recognition domains (CRD) that tightly bind to asparagines-linked high mannose glycans in viral enveloped glycoproteins on a broad spectrum of enveloped viruses in a calcium (Ca2)-dependent manner (T.B. Geijtenbeek et al., “Identification of different binding sites in the dendritic cell-specific receptor DC-SIGN for intercellular adhesion molecule 3 and HIV-1,” J. Biol. Chem. 277:11314-11320 (2002)). The C-type lectins therefore concentrate viruses on cells expressing DC-SIGN or L-SIGN, and facilitate binding and entry of viruses into cells.
It has been reported that DC-SIGN binds to HIV gp120 and facilitate HIV transmission to T cells (J. F. Arrighi et al., “DC-SIGN-mediated infectious synapse formation enhances X4 HIV-1 transmission from dendritic cells to T cells,” J Exp Med. 200(10):1279-88 (2004); T. B. Geijtenbeek et al., “Rhesus macaque and chimpanzee DC-SIGN act as HIV/SIV gp120 trans-receptors, similar to human DC-SIGN,” Immunol Lett. 79:101-7 (2001); M. Satomi et al., “Transmission of macrophage-tropic HIV-1 by breast-milk macrophages via DC-SIGN,” J. Infect. Dis. 191(2):174-81 (2005); E. J. Soilleux et al., “Placental expression of DC-SIGN may mediate intrauterine vertical transmission of HIV,” J. Pathol. 195:586-592 (2001)). DC-SIGN and L-SIGN have been shown to be high affinity binding rectors for hepatitis C virus glycoprotein E2 (P. Y. Lozach et al., “DC-SIGN and L-SIGN are high affinity binding receptors for hepatitis C virus glycoprotein E2,” J. Biol. Chem. 278(22):20358-66 (2003)), and mediate transinfection of liver cells by hepatitis C virus (Lozach et al., 2004, supra; Cormier et al., 2004, supra). DC-SIGN has also been found to mediate Dengue virus infection of human dendritic cells (Navarro-Sanchez et al., 2003, supra). Both DC-SIGN and L-SIGN have been shown to mediate cellular entry by Ebola virus in cis and in trans (Alvarez et al., 2002, supra; G. Simmons et al., “DC-SIGN and DC-SIGNR bind Ebola glycoproteins and enhance infection of macrophages and endothelial cells,” Virology 305(1):115-23 (2003)). In other reports, a broad spectrum of enveloped viruses including Retroviridae (human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV) and feline immunodeficiency virus (FIV)), Flaviviridae (Dengue virus, West Nile virus and hepatitis C virus (HCV)), Filoviridae (Ebola and Marburg virus), Coronaviridae (severe acute respiratory syndrome coronavirus (SARS-CoV)), Togaviridae (Sindbis virus) and Herpesviridae (human cytomegalovirus (human CMV)), has been reported to use DC-SIGN and/or L-SIGN as recognition and adhesion receptor for enhanced infection in vitro (P. Y. Lozach et al., “The C type lectins DC-SIGN and L-SIGN: receptors for viral glycoproteins,” Methods Mol. Biol. 379:51-68 (2007)).
DC-SIGN and L-SIGN are homotetrameric type II membrane proteins and can recognize a relatively large number of N-linked carbohydrates, such as mannose-containing glycoconjugates and fucose-containing Lewis bloodgroup antigen, on viral enveloped glycoproteins through a C-terminal carbohydrate recognition domain (D. A. Mitchell et al., “A novel mechanism of carbohydrate recognition by the C-type lectins DC-SIGN and DC-SIGNR. Subunit organization and binding to multivalent ligands,” J. Biol. Chem. 276:28939-28945 (2001); H. Feinberg et al., “Structural basis for selective recognition of oligosaccharides by DC-SIGN and DC-SIGNR,” Science 294:2163-2166 (2001)). Of the four glycoproteins on PRRSV virion envelope, GP2a, GP3, GP4 and GP5 contain 2 to 7 N-glycosylation sites, respectively, based on the computer prediction. Endoglycosidase treatment suggested that all putative sites are occupied by complex-type N-glycans (Meulenberg et al., 1998, supra). These observations suggest that DC-SIGN/L-SIGN may interact with one or more glycoproteins on PRRSV virion, thus mediating PRRSV entry and endocytosis. DC-SIGN is expressed on DCs and some types of macrophages, which are both important targets for PRRSV replication. L-SIGN was found to be expressed on sinusoidal endothelial cells and on placental macrophages. Placental expression of DC-SIGN was found to mediate intrauterine vertical transmission of HIV (Soilleux et al., 2001, supra). Coincidently, PRRSV is known to cause severe reproductive diseases in pregnant sows.
SARS-Coronavirus, belonging to the family Coronaviridae in the order Nidovirales together with the Arteriviridae family in which PRRSV is a member, was also shown to use the S glycoprotein to bind to DC-SIGN and L-SIGN during virus infection and pathogenesis (Marzi et al., 2004, supra; Z. Y. Yang et al., “pH-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through DC-SIGN,” J. Virol. 78(11):5642-50 (2004)). Although PRRSV and coronaviruses belong to the same super family, further testing is necessary to determine whether PRRSV will similarly use the DC-SIGN or L-SIGN for infection and pathogenesis.
A recent study reported that the Nipah virus surface glycoprotein protein (NiV-G) was able to bind to hLSECtin and hLSECtin was the putative receptor for Nipah virus surface glycoprotein protein (NiV-G) (T. A. Bowden et al., “Crystal Structure and Carbohydrate Analysis of Nipah Virus Attachment Glycoprotein: A Template for Antiviral and Vaccine Design,” J. Virol. in press 2008). The interaction was mediated by the GlcNAcβ1-2Man terminal structures in NiV-G. The envelope surface glycoproteins of Ebola virus (the truncated glycans) as well as the spike protein of severe acute respiratory syndrome coronavirus (SARS-CoV) bear these carbohydrate motifs and are uniquely recognized by hLSECtin (T. Gramberg et al., “LSECtin interacts with filovirus glycoproteins and the spike protein of SARS coronavirus,” Virology 340(2):224-36 (2005)). Unlike hDC-SIGN and hL-SIGN, the hLSECtin selectively bound to the glycoproteins terminating in the disaccharide GlcNAcβ1-2Man.
Furthermore, DC-SIGN and L-SIGN are considered two independent genes in the genomic level in human. Due to conserved sequences, they may have similar but distinct functions as shown in previous DC-SIGN/L-SIGN human studies. However, the biological or physiological role of L-SIGN is limited to the liver (mRNA of L-SIGN is only expressed in the liver) whereas DC-SIGN functions in the dendritic cells throughout the body.
Other related art has been published on human C-type lectin and human DC-SIGN. For instance, U.S. Pat. No. 6,190,886 (Hoppe et al.) describes a polypeptide comprising a collectin C-type lectin domain of human SP-D and the neck-region-lectin domain purified from lysates of bacterial cultures induced to express the recombinant proteins, wherein the polypeptide is able to trimerize in the collectin neck region. The suggested uses for the trimerized polypeptides are seeding collagen formation, as peptide-ligands for receptors, especially low-affinity binding (e.g., neuropeptides, interleukins), antigens, chemical compounds that are reactive upon activation, e.g., photo-activatable chemical crosslinkers, organic compounds such as caffeine and morphine, low affinity binding domains especially for the screening of potential inhibitors in pharmaceutical research, etc.
U.S. Pat. No. 6,455,683 (Yang et al.) describes isolated cDNA sequences encoding a human C-type lectin and three homologues referred to as “CLAX” (C-type Lectin, Activation Expressed) proteins. The patent discloses methods of using the nucleic acid sequences, polypeptides, fusion proteins having all or a portion (e.g., an extracellular region) of the human CLAX proteins, antibodies specific for the CLAXs, ligands and inhibitors for the human CLAXs. It is suggested that pharmaceutical compositions containing the proteins are used for the prevention and treatment of infectious, inflammatory and allergic diseases.
U.S. Pat. No. 6,280,953 (Messier et al.) provides methods for identifying polynucleotide and polypeptide sequences in human and/or non-human primates which may be associated with a physiological condition, such as disease including susceptibility (human) or resistance (chimpanzee) to development of AIDS. The physiological trait includes resistance to the progression of AIDS; the polynucleotide may be a human DC-SIGN gene; and the modulated function is then increased resistance to the progression of AIDS. It is suggested that the sequences are useful as host therapeutic targets and/or in screening assays.
U.S. Pat. No. 6,365,156 (Lee) relates to methods of increasing the half-life of a viral-specific ligand to be administered on a mucosal membrane wherein said membrane is colonized with bacteria, such as Lactobacillus, Streptococcus, Staphylococcus, Lactococcus, Bacteriodes, Bacillus, and Neisseria, by modifying the bacterial-specific ligand to bind the bacteria colonized on the mucosal membrane. The patent also discloses a chimeric molecule comprising a viral-specific ligand such as CD4, DC-SIGN, ICAM-1, HveA, HveC, poliovirus receptor, vitronectin receptor, CD21, or IgA receptor sequences and a bacterial-specific ligand such as an antibody, a peptide, a polypeptide, a protein or a carbohydrate.
U.S. Pat. No. 6,391,567 (Littman et al.) concerns human DC-SIGN as a receptor that is specifically expressed on dendritic cells and facilitates infection of T lymphocytes with Human Immunodeficiency Virus (HIV). The patent provides assays for identifying compounds that modulate the interaction of DC-SIGN and HIV and/or T cells and macrophage wherein the compounds inhibit the trans-enhancement of HIV entry into a cell.
U.S. Pat. No. 7,148,329 (Figdor et al.) deals with the use of mannose, fucose, plant lectins, antibiotics, proteins or antibodies against C-type lectins, that binds to a C-type lectin on the surface of a dendritic cell, in the preparation of a composition for modulating the immune response by modulating the adhesion of C-type lectin receptors on the surface of dendritic cells to the ICAM-receptors on the surface of T cells. The patent discloses antibodies that inhibit binding between dendritic cells and T-cells, that is, between DC-SIGN on the surface of a dendritic cell and an ICAM-3 receptor on the surface of a T-cell. The compositions are proposed for preventing/inhibiting immune responses to specific antigens, for inducing tolerance, for immunotherapy, for immunosuppression, for the treatment of auto-immune diseases, the treatment of allergy, and/or for inhibiting HIV infection.
As noted above, there is a biological relationship between DC-SIGN and ICAM-3 as part of an immunological superfamily. The intercellular adhesion molecules (ICAMs) are type I transmembrane glycoproteins belonging to a subfamily in the immunoglobulin (Ig) superfamily. Thus far, five members of the ICAM family (ICAMs 1-5) have been identified in mammals (C. G. Gahmberg et al., “Leukocyte adhesion--structure and function of human leukocyte beta2-integrins and their cellular ligands,” Eur. J. Biochem. 245:215-232 (1997)). They share functional and structural Ig-like domains and mediate cell-to-cell adhesion interactions relevant for the function of the immune system (T. A. Springer, “Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm,” Cell 76:301-314 (1994)). Except for ICAM-5, all other ICAM members bind to the integrin LFA-1 (CD11a/CD18) but showing large variation in tissue distributions (Gahmberg et al., 1997, supra). These adhesive interactions play important roles in mediating leukocyte trafficking through inflamed and uninflamed tissues and contribute to antigen-specific T-cell response. Of the ICAM members, ICAM-3 is thought to be the dominant ligand for LFA-1 during the initiation of the immune response, since both ICAM-1 and ICAM-2 are not expressed, or expressed at a very low level, on resting leukocytes and antigen-presenting cells (APC) (A. R. de Fougerolles et al., “Cloning and expression of intercellular adhesion molecule 3 reveals strong homology to other immunoglobulin family counter-receptors for lymphocyte function-associated antigen 1,” J. Exp. Med. 177:1187-1192 (1993)). The binding of ICAM-2 and ICAM-3 to the C-type lectin, human DC-SIGN, has been reported in that interaction of ICAM-3 with DC-SIGN establishes initial contact between dendritic cells and resting T-cells during antigen presentation whereas binding of ICAM-2 to human DC-SIGN regulates emigration of dendritic cells and transmigration through endothelium (T. B. Geijtenbeek et al., “Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3 receptor that supports primary immune responses,” Cell 100:575-585 (2000); T. B. Geijtenbeek et al., “DC-SIGN-ICAM-2 interaction mediates dendritic cell trafficking,” Nat. Immunol. 1:353-357 (2000)).
Full-length ICAM molecules contain a signal peptide sequence, two (ICAM2 and ICAM4), five (ICAM1 and ICAM3) or nine (ICAM-5) extracellular Ig-like domains, a hydrophobic transmembrane domain (TMD), and a cytoplasmic tail (CT). Each Ig-like domain is encoded by a distinct exon (G. Voraberger et al., “Cloning of the human gene for intercellular adhesion molecule 1 and analysis of its 5′-regulatory region. Induction by cytokines and phorbol ester,” J. Immunol. 147:2777-2786 (1991); C. M. Ballantyne et al., “Characterization of the murine Icam-1 gene,” Genomics 14:1076-1080 (1992)). Isoforms of murine ICAM-1 generated by alternative splicing have been identified in ICAM-1-deficient mice (P.D. King et al., “Novel isoforms of murine intercellular adhesion molecule-1 generated by alternative RNA splicing,” J. Immunol. 154:6080-6093 (1995); N.K. van Den Engel et al., “Circulating forms of intercellular adhesion molecule (ICAM)-1 in mice lacking membranous ICAM-1,” Blood 95:1350-1355 (2000)). Each murine ICAM-1 isoform is generated from the complete skipping of exons encoding Ig-like domains 2, 3, and/or 4. In addition, the existence of an alternative 5′ splice site in exon 6 also yields a murine ICAM-1 isoform with a 69-nt deletion from the 3′-terminus of exon 6 (J. P. Mizgerd et al., “Exon truncation by alternative splicing of murine ICAM-1,” Physiol. Genomics 12:47-51 (2002)). In murine ICAM-4, a transmembrane-domain-lacking isoform causing by intron retention was also identified (G. Lee et al., “Novel secreted isoform of adhesion molecule ICAM-4: potential regulator of membrane-associated ICAM-4 interactions,” Blood 101:1790-1797 (2003)). All the ICAM isoforms identified to date are fully functional, indicating that alternative mRNA splicing plays distinct roles in different immune response pathways.
Two comparative sequence analysis studies based on human-pig-mouse-rat or human-dog-mouse-rat genomic regions revealed that the ICAM3 gene has been lost in the rodent genome (H. Sugino, “ICAM-3, a ligand for DC-SIGN, was duplicated from ICAM-1 in mammalian evolution, but was lost in the rodent genome,” FEBS Lett. 579:2901-2906 (2005); T. Leeb and M. Muller, “Comparative human-mouse-rat sequence analysis of the ICAM gene cluster on HSA 19p13.2 and a 185-kb porcine region from SSC 2q,” Gene 343:239-244 (2004)). The organization of ICAM3 genes in human, non-human primates and bovine is similar, which contains seven putative exons, and exons 3 to 7 are clustered at the 3′-proximal region of the gene (P. Kilgannon et al., “Mapping of the ICAM-5 (telencephalin) gene, a neuronal member of the ICAM family, to a location between ICAM-1 and ICAM-3 on human chromosome 19p13.2,” Genomics 54:328-330 (1998); E. K. Lee et al., “Cloning and sequencing of a cDNA encoding bovine intercellular adhesion molecule 3 (ICAM-3),” Gene 174:311-313 (1996)). For porcine ICAM-3, the gene sequence is not yet completely known since only the region from exon 1 to partial exon 5 has been identified and sequenced (Leeb and Muller, 2004, supra). In addition, the cDNA of porcine ICAM-3 has not been identified thus far.
Nonsense mutations falling within an exon can induce exon skipping during the pre-mRNA splicing process, which is designated as nonsense-associated altered splicing (NAS) (L. Cartegni et al., “Listening to silence and understanding nonsense: exonic mutations that affect splicing,” Nat. Rev. Genet. 3:285-298 (2002); L.E. Maquat, “The power of point mutations,” Nat. Genet. 27:5-6 (2001); H. C. Dietz et al., “The skipping of constitutive exons in vivo induced by nonsense mutations,” Science 259:680-683 (1993)). NAS is usually disease-associated, as has been shown in a few disease-causing genes (Cartegni et al., 2002, supra), since premature termination of translation would result in failing to produce a functional protein. The mechanisms of NAS are believed to be due to the occurrence of a translation-like nucleus scanning before slicing, indirect nonsense-mediated mRNA decay (NMD) or exonic splicing enhancer (ESE) disruption (id.).
While human DC-SIGN is involved in the transmission of various enveloped viruses such as human immunodeficiency virus, hepatitis C virus, Dengue virus and SARS-Coronavirus to their respective target cells, the characteristics and properties of DC-SIGN proteins obtained from other species have not been shown to mimic hDC-SIGN as a rule. Therefore, further testing is necessary to allocate the function of any given DC-SIGN. Before the current discovery, the DC-SIGN and other LSECtin related homologues from the pig species had not yet been isolated, identified or characterized.
It is therefore an important object of the present invention to obtain the cloning and characterization of the full nucleic acid molecule encoding new porcine DC-SIGN and porcine LSECtin proteins heretofore not described in the pig genome database.
It is another important object of the invention to identify and characterize the complete nucleic acid molecules encoding new porcine ICAM-3 isoforms from in vitro cultured porcine monocyte-derived dendritic cells.
It is an additionally significant object of the invention to use pDC-SIGN, pLSECtin, pICAM-3 alone or in certain combinations as fused proteins with hDC-SIGN, hL-SIGN or hLSECtin in a new method for propagating viruses, particularly enveloped viruses with an emphasis on porcine enveloped viruses, making use of new transfected cells or cell lines stably expressing pDC-SIGN, pLSECtin and/or pICAM-3.
It is a further object of the invention to raise an antibody that specifically binds to an amino acid sequence of the pDC-SIGN protein and is utilizable to enhance the immunogenic activity of poor antigenic substances. Raising an antibody that specifically binds to an amino acid sequence of the pLSECtin and pICAM-3 proteins is also highly desirable.
Further purposes and objects of the present invention will appear as the specification proceeds.
The foregoing objects are accomplished by providing and isolating the new and complete nucleic acid sequences encoding pDC-SIGN, pICAM-3 and pLSECtin, using the nucleotide sequences encoding the proteins in specially designed vectors to propagate enveloped viruses, raising antibodies and the like.